Our group has been exploring the mechanisms by which chaperones and proteases act together to regulate protein activity and degradation. More specifically, we are investigating protein remodeling by the E. coli ClpA and ClpX ATP-dependent molecular chaperones and the role of these chaperones in protein degradation by ClpAP and ClpXP. The mechanism of protein recognition by ClpA has been studied using a high affinity substrate, RepA, which is activated for DNA binding by ClpA and degraded by ClpAP. By characterizing RepA derivatives with N- or C-terminal deletions, we found that the N-terminal portion of RepA is required for recognition. RepA derivatives lacking the N-terminal 5 or 10 amino acids are degraded by ClpAP at a rate similar to full-length RepA, while RepA derivatives lacking 15 or 20 amino acids are degraded much more slowly. We constructed fusions of RepA and green fluorescent protein (GFP), a protein not recognized by ClpA, and found that a fusion containing the N-terminal 15 amino acids of RepA was degraded by ClpAP. Thus, the first 15 amino acids of RepA are necessary and sufficient to target the fusion protein for degradation by ClpAP. The RepA-GFP fusion has also been used to investigate the mechanism of protein remodeling by ClpA and translocation to ClpP. Substrate binding alone is not sufficient to destabilize the native structure of the GFP portion of the fusion protein. However, in the presence of ATP, ClpA catalyzes unfolding of the RepA-GFP fusion protein and can sequester the unfolded intermediate if a non-hydrolyzable analog is added to displace ATP. We found that although ClpA is unable to recognize native proteins lacking recognition signals, including GFP and rhodanese, it interacts with those same proteins when they are unfolded. Degradation of unfolded untagged proteins by ClpAP requires ATP even though the initial ATP-dependent unfolding reaction is bypassed. These results suggest that there are two ATP-requiring steps: an initial protein unfolding step followed by translocation of the unfolded protein to ClpP. Another important question we are addressing is how regulatory proteins act to target specific substrates for degradation. We are studying the regulation of degradation of the E. coli stationary phase sigma factor, sigma S, by a response regulator protein, RssB, in conjunction with the ClpXP protease. In vitro, ClpXP alone degrades sigma S poorly and the addition of RssB greatly stimulates the reaction. ATP is essential and acetyl phosphate, which phosphorylates RssB, accelerates the rate of the reaction. RssB specifically stimulates degradation of sigma S by ClpXP; it does not stimulate degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP. RssB and sigma S assemble into a stable complex in the presence of acetyl phosphate and form a tertiary complex with ClpX. A larger sigma S-RssB-ClpXP complex assembles in the presence of ClpP. The addition of ATP promotes sigma S degradation and the release of RssB from ClpXP. RssB participates in multiple rounds of sigma S degradation, demonstrating the catalytic role of RssB. Our results suggest that a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.